Project Summary. The heterotrimeric G protein G?q regulates platelet activation, blood pressure and cardiac function. While G?q is best known for its ability to stimulate phospholipase Cp (PLC|3), it also binds to G protein-coupled receptor kinase 2 (GRK2), which competitively inhibits PLCp activation, and to p63RhoGEF, a Rho guanine nucleotide exchange factor that modulates cytoskeletal structure. We recently demonstrated that a functional Gaq chimera could be produced in amounts sufficient for the structure determination of the G?q -GRK2-Gpy complex. The surprising arrangement of activated heterotrimeric G proteins in this assembly suggested that RGS proteins and receptors could also associate to form even higher order signaling complexes. Aim 1 examines the ability of RGS proteins such as RGS2 to bind GRK2- bound G?q using a flow-cytometry binding assay, size-exclusion chromatography and crystallographic studies of Gaq-RGS and RGS- G?q GRK2-Gpy complexes. Aim 2 investigates changes in the orientation of activated G?q at the membrane upon effector binding as well as the interactions of G?q -GRK2-Gpv and RGS proteins with intact receptors or their cytoplasmic loops in a membrane environment. The G?q chimera also opens the door to the structural analysis of other G?q -effector interactions. To initiate these efforts, Aim 3 seeks to define the molecular basis for G?q -mediated activation of p63RhoGEF through site-directed mutagenesis, fluorescence polarization nucleotide exchange assays and structural studies. Relevance. By focusing our proposal on two unique effectors of G?q, GRK2 and p63RhoGEF, we seek to define general paradigms for heterotrimeric G protein signaling through G?q. We have also focused on G?q, GRK2, RGS2 and p63RhoGEF because all are strongly linked to cardiovascular physiology and disease, and it is not unreasonable to expect that these proteins coordinate their activities, either directly or indirectly, in living cells. GRK2 and G?q /n are essential for proper heart development and function, RGS2 regulates blood pressure via attenuation of G?q signaling, and p63RhoGEF induces changes in myocytes that are characteristic of cardiac hypertrophy. [unreadable] [unreadable] [unreadable]